Click-iT® Labeling and Detection Tools

If our ready-to-use kits do not match your experimental requirements, a range of Click-iT® tools are available to allow you to develop a labeling and detection strategy that addresses your particular biological questions. In the labeling tools section you will find the primary building blocks for incorporating Click-iT® labels into the proteins or nucleic acids in your biological model. The table of detection tools shows the complementary probes offered with fluorescent tags that provide visualization or capture of your target molecule.

Detection tools

Standard reactions

Azides and alkynes can be used interchangeably in standard click reactions, so either one can serve as the tagged substrate or be labeled for the detection step. Some biomolecules are sensitive to the amount of copper used to catalyze standard click reactions. In particular, proteins such as GFP and R-PE exhibit reduced fluorescence and nucleic acids can be denatured by a high-copper environment.

Copper sensitivity

Biomolecules exhibit varying sensitivity to copper concentration. The high copper concentration used to catalyze traditional click reactions maintains the fluorescence of APC, but not GFP. At medium copper concentrations, GFP and APC fluorescence is maintained, but phalloidin binding is lost (Table 1). With Click-iT® Plus reagents, the copper concentration can be optimized to preserve the signal in your fluorescent proteins and also drive the EdU click reaction.

Table 1. The effects of copper on fluorescence and phalloidin binding.

Copper concentration

Maintained

Lost

High

APC fluorescence

GFP fluorescence

Phalloidin binding

Medium

APC fluorescence

GFP fluorescence

Phalloidin binding

Low

APC fluorescence

GFP fluorescence

Phalloidin binding

Picolyl azides for low-copper reactions

Click-iT® Plus reagents enable researchers to perform copper-catalyzed click reactions with compounds that are sensitive to copper, while retaining all of the benefits of the original azide/alkyne click reaction. Each Click-iT® Plus picolyl azide–based tool box includes everything required to optimize the copper concentration and protect against undesired copper side reactions with proteins (e.g., GFP, RPE), nucleic acids (e.g., RNA, oligos), and even small molecules (e.g., phalloidin).

Additionally the Click-iT® Plus picolyl azides can be used to increase sensitivity or accelerate the reactions by using the original copper-catalyzed reaction conditions.

DIBO alkynes for copper-free reactions

DIBO reagents can be used to perform click reactions with azide-modified targets without the use of heavy metal catalysis. DIBO reactions are ideal for surface labeling of live cells and also minimize damage to fluorescent proteins like GFP or R-PE. For intracellular labeling, the nonspecific binding of DIBO alkynes creates a background signal, and a labeling strategy using picolyl azides is the preferred solution in these instances.